519-529.qxd 20/12/2010 01:05 ÌÌ ™ÂÏ›‰·519 ONCOLOGY REPORTS 25: 519-529, 2011 519 Screening of novel immunostimulatory CpG ODNs and their anti-leukemic effects as immunoadjuvants of tumor vaccines in murine acute lymphoblastic leukemia JIN WANG, WANGGANG ZHANG, AILI HE, WANHONG ZHAO and XINGMEI CAO Department of Hematology, Second Affiliated Hospital, Medical School of Xi'an Jiaotong University, Xi'an 710004, Shaanxi Province, P.R. China Received July 29, 2010; Accepted October 11, 2010 DOI: 10.3892/or.2010.1093 Abstract. Acute lymphoblastic leukemia (ALL) is a common Additional therapeutic strategies are required in order to malignant disease and a major cause of mortality due to further prolong remission duration and eradicate minimal recurrent disease. Immunotherapy is a promising strategy for residual disease, ideally with less toxicity than conventional eradicating minimal residual disease and thus preventing the chemotherapy. One of the these approaches is active immuno- relapse of leukemia. Apart from stem cell transplantation, therapy with novel vaccination regimens. We previously CpG oligodeoxynucleotides (ODNs) are excellent candidates launched a phase-I clinical trial and evaluated the efficacy for the immunotherapy of leukemia. However, the number of and toxicity of vaccination in patients with relapsed or usable CpG ODNs is limited. In this study, we tested a panel refractory acute leukemia, which proved to be a feasible, of CpG ODNs and obtained three CpG ODN sequences with safe, and capable way of eliciting anti-leukemic responses strong immunostimulatory activity by comparing their in vivo (1). capacity to activate lymphocytes. The data revealed that the As tumor cells are considered to be poorly immunogenic, flanking bases, the spacing of individual CpG motifs and poly- it is difficult to elicit an effective specific anti-tumor activity guanosine ends, contribute to the immunostimulatory activity from these cells alone. In addition, tumor cells develop of a CpG ODN. In the immunotherapy of murine leukemia various immune mechanisms to escape from host immune with the novel ODN as the adjuvant, we found that CpG Seqs surveillance, for example, down-regulating co-stimulatory 14 and 19 were effective in the treatment of a leukemia model signals essential for the activation of the immune system. An by prolonging survival span, augmenting natural killer cell appropriate adjuvant could help break the immune tolerance and CTL cytotoxicity, as well as increasing the number of in acute leukemia patients. To that end, tumor antigens need long-term survivors. The ability of CpG ODN to induce both to be administered in combination with adjuvants. The most strong innate and adaptive anti-leukemic immune activity common adjuvant used so far in experimental models is could render it an appropriate agent for therapeutic applica- Incomplete Freund's adjuvant (IFA). IFA has also been tions in acute leukemia. This study demonstrates the feasibility successfully used in human immunotherapy against melanoma of active immunotherapy with CpG ODNs in patients with involving gp 100 peptide immunization (2). However, it is acute leukemia and thus represents a potential alternative not widely used in human vaccination protocols due to its therapeutic strategy for eradicating residual disease which is undesirable severe side-effects, which include swelling and resistant to conventional cytoreductive treatment. indurations at the injection site. IFA has also been reported to induce specific CTL tolerance as opposed to immunity against Introduction immunizing antigens (3). Cytokines, such as interleukin (IL)-2, are another type of adjuvant being increasingly used Despite intensive chemotherapy regimens, long-term survival in hematological malignant disease. However, systemic occurs in only a third of cases of adult acute leukemia. therapy with cytokines is also associated with severe toxic side effects, including wheezing and difficulty in breathing, dizziness when changing position, rapid weight gain or sudden _________________________________________ swelling, chest pain or irregular heartbeat, and so on. For these reasons, alternative potent and safe adjuvants need to be identified. Correspondence to: Dr Wanggang Zhang, Department of Hematology, Second Affiliated Hospital Medical School of Xi'an In the last few years, a type of synthetic oligonucleotide Jiaotong University, 157 Xiwu Road, Xincheng District, Xi'an containing unmethylated cytosine and guanine dinucleotides 710004, Shaanxi Province, P.R. China within specific-sequence contexts (CpG motifs), has been E-mail: [email protected] shown to provoke strong immune responses (4) as well as remarkable T cell-dependent anti-tumor efficacy in a series Key words: CpG oligodeoxynucleotides, toll-like receptor, of murine tumor models. CpG oligodeoxynucleotides (ODNs) immunotherapy, tumor vaccine, acute leukemia are thought to mimic bacterial DNA and are recognized as a ‘danger’ signal by the innate immune system of mammals. 519-529.qxd 20/12/2010 01:05 ÌÌ ™ÂÏ›‰·520 520 WANG et al: SCREENING OF NOVEL CpG ODNs AND THEIR ANTI-LEUKEMIC EFFECTS As toll-like receptor (TLR)9 agonists, CpG ODNs boost innate The sequences of CpG ODN used in this study are described immunocytes such as B-cells, dendritic and natural killer in Table I. (NK) cells, as well as monocytes, causing them to proliferate, mature and produce various cytokines, including IL-6, and -12, The in vitro study tumor necrosis factor (TNF) and interferon (5). In addition, Cell preparation and culture. Human peripheral blood CpG ODN stimulation leads to the generation of Th1 type mononuclear cells (PBMCs) were collected and isolated from adaptive immune responses (6-8). These properties have led the peripheral blood of healthy volunteers by Ficoll-Paque to considerable interest in the clinical use of CpG ODNs in density gradient centrifugation (Histopaque-1077, Sigma- vaccines, cancer and autoimmune disease therapy (9). Aldrich, St. Louis, MO, USA) as previously described (16), Despite these promising benefits of CpG ODNs, the after receiving approval from the Institute for Cancer numbers of usable CpG ODNs are limited in the current Research, College of Life Science and Technology, Xi'an clinical setting of cancer immunotherapeutics. The clinical Jiaotong University. Cells were suspended in RPMI-1640 restriction of CpG ODNs is due to several reasons: The culture medium supplemented with 10% (v/v) heat-inactivated absence of convenient screening assays, difficulty to predict (56˚C, 30 min) fetal bovine serum, 100 U/ml penicillin, and the structure of potent CpG ODNs and the differences 100 μg/ml streptomycin (all from Life Technologies, Grand between species of CpG motif recognition (10). While the Island, NY). All compounds were purchased endotoxin- primary focus for the medicinal application of CpG ODNs tested. Viability was determined before and after incubation has been on cancer immunotherapy in pre-clinical and clinical with ODN by trypan blue exclusion (conventional micro- trials against cancer (11-14), there have been no reports of scopy). In all experiments, >95% of PBMCs were viable. the in vivo consequences of the immunotherapeutic effects of The cells (final concentration, 2.5x106 cells/ml) were then CpG ODN treatment as adjuvants in acute leukemia. In this cultured in complete medium in a 5% CO2-humidified study, in order to develop CpG ODNs for clinical use, we incubator at 37˚C for 48 h. Different ODNs (Table I; final designed a panel of CpG ODNs (Table I) with different concentration, 1 μg/ml) and LPS (from Salmonella structural features following the characteristics which typhimurium; Sigma Aldrich) were used as stimuli. contribute to the potency of a human CpG ODN (15). We Spleens were removed from 6-week-old female Balb/c then compared their ability to induce cell proliferation and mice, dispersed and centrifuged at 1,000 rpm for 5 min. The cytokine secretion, and selected the most potent ODN. supernatant was removed and the cell pellet was re-suspended Finally, we observed the treatment effect of vaccines with the in 0.84% NH4Cl to eliminate red blood cells. Cells were formulation of a CpG ODN adjuvant and their ability to elicit washed twice with RPMI-1640 medium, re-suspended in the anti-leukemic immune responses in a mouse model. culture medium and cultured at 5x106/ml with the different ODNs. Materials and methods Cell proliferation assay. The proliferation of human PBMCs Mice. Six-week-old female Balb/c mice were purchased from and mice splenocytes was assessed by the quantitative assay, the Laboratory Animal Center, Medical Shcool of Xi'an Cell Counting Kit-8 (CCK-8) (Dojindo, Gaithersburg, MD) Jiaotong University. Female DBA/2 mice between 4 and 6 according to the manufacturer's instructions. Briefly, this weeks of age were purchased from the Shanghai Laboratory cell-counting method is an optical density colorimetric assay Animal Center after approval by the Chinese Academy of that quantifies the number of viable cells per well based on Sciences. The animals were housed in a specific pathogen- the activity of cellular dehydrogenases. At 44 h, 20 μl/well free animal facility under isothermal conditions and were CCK-8 were added to the medium and cultured together for allowed access to food and water ad libitum. 4 h, and then the absorbance at 450 nm was determined. Leukemia cell line and cell culture. L1210, which is a murine Detection of cytokines. PBMCs and mouse splenocytes were lymphoid leukemia cell line of DBA/2 origin, was a generous plated in 24-well dishes using 2.5x106 or 5x106 cells/ml, gift from Professor Yili Wang (Institute for Cancer Research, respectively. The CpG ODNs were added to the cell cultures College of Life Science and Technology, Xi'an Jiaotong at a final concentration of 1 μg/ml. The cells were then incu- University, China) and was maintained in RPMI-1640 (Gibco, bated at 37˚C for 24 h and the supernatants were collected for BRL, Gaithersburg, MD, USA) supplemented with 10% ELISA assays. The experiments were performed twice for heat-inactivated fetal bovine serum, 100 U/ml penicillin, and each CpG ODN in triplicate.